SNP markers, detection methods and applications related to muscle shear force on pig chromosome 8

Abstract

This invention discloses a SNP marker related to muscle shear force on pig chromosome 8, along with its detection method and application. The SNP marker is located on pig chromosome 8 at nucleotide rs332282301 of the international pig genome version 11.1 reference sequence, exhibiting A / G polymorphism. This SNP marker is highly significantly correlated with pig muscle shear force. A primer pair for detecting the SNP marker is provided, with the upstream primer being SEQ ID NO: 2 and the downstream primer being SEQ ID NO: 3. The SNP marker provided by this invention is related to the tenderness of pigs. Identifying this SNP marker can screen pigs with low muscle shear force (better tenderness), and establishing a pig population with low muscle shear force (better tenderness) has significant economic and social value.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology and relates to SNP markers and detection methods and applications related to muscle shear force on pig chromosome 8. Background Technology

[0002] As people's living standards continue to improve, their demand for pork is increasing, and their requirements for pork quality are also rising. Meat quality characteristics are indicators for evaluating pork quality, and these mainly include intramuscular fat, tenderness, drip loss, pH, and meat color. Pork tenderness refers to the feeling of pork when eaten after cooking. Many factors can affect pork tenderness, mainly including the structure and type of proteins in the muscle, the muscle location, and fiber diameter. Generally, the shear force of cooked pork is used to measure its tenderness. Shear force reflects the resistance when cutting pork; the greater the shear force, the tougher the meat and the less tender it is; the smaller the shear force, the more tender the meat and the better the taste.

[0003] Because in vivo shear force in pork cannot be measured, and the cost of measurement is high, traditional breeding methods struggle to efficiently improve shear force. This study used phenotypic determination and gene chip analysis of pig muscle shear force, employing genome-wide association analysis to identify candidate genes or molecular markers influencing pig shear force, providing key molecular markers for marker-assisted breeding and genome-wide selection of pig shear force. Summary of the Invention

[0004] The purpose of this invention is to address the problems of time-consuming and labor-intensive traditional breeding of pork shear force, difficulties in phenotypic determination, and slow breeding results, and to provide breeding molecular markers developed from SNP markers related to pork shear force.

[0005] Another object of the present invention is to provide primers and detection methods for detecting the above-mentioned SNP markers.

[0006] Another object of the present invention is to provide the use of the above-mentioned SNP marker.

[0007] A single nucleotide polymorphism (SNP) marker associated with porcine muscle shear force is disclosed. The SNP marker is located on porcine chromosome 8 at nucleotide locus rs332282301 of the international porcine genome version 11.1 reference sequence and exhibits A / G polymorphism. This SNP marker is highly significantly correlated with porcine tenderness. Porcines with the GG genotype at rs332282301 exhibit significantly lower shear force than those with the AA and AG genotypes.

[0008] A method for developing molecular markers based on the SNPs described in this invention involves designing primer pairs using nucleotide sequences containing the SNP markers described in this invention as base sequences, and performing PCR amplification using porcine genomic DNA as a template to convert the SNP markers described in this invention into molecular markers.

[0009] The primer pair sequence is: upstream primer: SEQ ID NO: 2, downstream primer: SEQ ID NO: 3; the molecular marker sequence is shown in SEQ ID NO: 1, and the SNP site is located at position 301, exhibiting A / G polymorphism.

[0010] Molecular markers obtained according to the method described above in this invention.

[0011] The preferred sequence of the molecular marker is shown in SEQ ID NO: 1, and the SNP site is located at position 301, exhibiting A / G polymorphism.

[0012] A primer pair for detecting the SNP marker, wherein the upstream primer is SEQ ID NO: 2 and the downstream primer is SEQ ID NO: 3.

[0013] A method for detecting the SNP marker described in this invention includes PCR amplification of a sequence containing the SNP marker described in this invention in the pig genome, sequencing the amplification product, and determining the A / G polymorphism of the site.

[0014] The method for detecting the SNP markers described in this invention includes the following steps:

[0015] (1) Take muscle tissue samples from pigs and extract total DNA;

[0016] (2) Using the extracted porcine genomic DNA as a template, PCR amplification was performed using the primers described above;

[0017] (3) Sequencing of the amplified product, analysis of the sequencing results, and interpretation of the A / G polymorphism at position 301 of SEQ ID NO: 1.

[0018] The application of the molecular markers described in this invention in screening populations with low muscle shear force or specialized strains.

[0019] The application of the primer pairs described in this invention in screening populations with low muscle shear force or specialized strains.

[0020] A method for screening low shear force populations of pigs includes detecting the genotype of the rs332282301 nucleotide site on chromosome 8 of the pig international pig genome version 11.1 reference sequence, and selecting individuals of the GG and AG genotypes at the rs332282301 nucleotide site as priority for breeding replacement pigs.

[0021] As a preferred embodiment of the present invention, the pig breed used is the Large White pig.

[0022] As a preferred embodiment of the present invention, the method for detecting the genotype at the rs332282301 nucleotide site on chromosome 8 of the pig international pig genome version 11.1 reference sequence is selected from PCR or gene sequencing.

[0023] Beneficial effects

[0024] The SNP marker provided by this invention is related to the shear force (tenderness) of pork. Therefore, by identifying this SNP marker, pigs with lower shear force can be screened. The establishment of a low muscle shear force (better tenderness) pig population has significant economic and social value. Attached Figure Description

[0025] Figure 1 The distribution of SNP marker-logP values ​​on chromosomes was obtained for genome-wide association analysis of muscle shear force in pigs. Information on the 18 autosomes of pigs is represented on the X-axis.

[0026] Figure 2 Electrophoresis image of the rs332282301 gene locus fragment amplified using the primers of this invention.

[0027] Figure 3 Figure 1 shows the DNA sequencing results of different genotypes at the rs332282301 mutation site, where Figure A represents the AA genotype, Figure B represents the AG genotype, and Figure C represents the GG genotype. Detailed Implementation Plan

[0028] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0029] Example

[0030] 1. Experimental animals

[0031] 413 pigs were sampled and their muscle tissue was taken.

[0032] 2. Shear force determination

[0033] The method for determining the shear force of pork refers to the national standard "NY / T 1180-2006 Determination of Meat Tenderness - Shear Force Test Method". The shear force of pork is determined using a digital display muscle tenderness meter (C-LM3B type). Each individual is measured three times and the average value is taken. The unit is cattle (N).

[0034] 3. Extraction of pig genomic DNA

[0035] One muscle tissue sample was collected from 413 pigs for individual DNA extraction.

[0036] Referring to the instructions for the Tissue DNA Extraction Kit from Tiangen Biotech Co., Ltd., the extraction steps are as follows:

[0037] ① First, add 68 mL of buffer GD and 200 mL of anhydrous ethanol to the wash buffer PW, and mix thoroughly.

[0038] ② Collect approximately 100 mg of ear tissue sample and place it in a 2 mL EP tube. After completely cutting it into small pieces, add 200 μL of buffer GA and shake until completely suspended.

[0039] ③ Add 20 μL of proteinase K solution, mix well, and place in a 56°C metal bath for digestion overnight until the tissue sample dissolves. Briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0040] ④ Add 200 μL of buffer GB, mix thoroughly by inverting, place in a 70℃ metal bath for 10 min, the solution should become clear, and briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0041] ⑤ Add 200 μL of anhydrous ethanol and shake thoroughly for 15 seconds. At this time, flocculent precipitate may appear. Briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0042] ⑥ Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3, place the adsorption column in the collection tube, then centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and put the adsorption column CB3 back into the collection tube.

[0043] ⑦ Add 500 μL of buffer GD to the adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and place the adsorption column CB3 into the collection tube.

[0044] ⑧ Add 600 μL of washing buffer PW to the adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and place the adsorption column CB3 into the collection tube.

[0045] ⑨ Repeat step ⑧.

[0046] ⑩ Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm for 2 minutes, and discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to thoroughly dry any residual washing liquid in the adsorption material.

[0047] Transfer the adsorption column CB3 into a clean centrifuge tube. Add 100 μL of elution buffer TE to the middle of the adsorption membrane. Incubate at room temperature for 2-5 min, then centrifuge at 12,000 rpm for 2 min. Collect the solution in the centrifuge tube. Add the centrifuged solution back to the adsorption column CB3. Incubate at room temperature for 2 min, then centrifuge at 12,000 rpm for 2 min. Collect the solution in the centrifuge tube.

[0048] The quality and concentration of DNA were determined using a Nanodrop-2000 spectrophotometer. All DNA concentrations were diluted to 50 ng / μL and stored at -20°C for later use.

[0049] 4. Target fragment PCR amplification and sequencing

[0050] PCR amplification was performed using porcine genomic DNA as a template. The reaction system included 1 μL of DNA template, 1 μL each of the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, 9.5 μL of ddH2O, and 12.5 μL of PCR mix. The amplification program was as follows:

[0051]

[0052]

[0053] The amplified product was subjected to agarose gel electrophoresis. The product fragment size was approximately 458 bp. The electrophoresis results are as follows: Figure 2 As shown. The remaining amplification products were sequenced, and the sequencing results were compared and verified for accuracy using DNAman software. The rs332282301 site was genotyped using Chromas software.

[0054] 5. Statistical Analysis

[0055] Association analysis between genotype and phenotype was performed using a general linear model in SAS 9.4 software, as follows: Yijk=μi+Bj+Gk+ejk

[0056] Where Yijk is the shear force of the individual; μ i B represents the mean shear force of the population. j Represents the fixed effects of slaughter batches; G k For the fixed effect of SNP labeling; e jk It is a residual.

[0057] 6 Results

[0058] Table 1 shows the effects of different genotypes at the rs332282301 locus on shear force in pig muscle. The results indicate that there were significant differences in shear force among the three genotypes at the rs332282301 locus (P<0.05). Specifically, the shear force of individuals with the GG and AG genotypes was significantly lower than that of individuals with the AA genotype (P<0.05), and the shear force of individuals with the GG genotype was significantly lower than that of individuals with the AG genotype (P<0.05). Therefore, selective breeding for individuals with the GG and AG genotypes at the rs332282301 locus in pig successive generations is beneficial for reducing the shear force in the pig population, thereby improving the tenderness of the pork.

[0059] Table 1. Association analysis between the rs332282301 locus on porcine chromosome 8 and shear force in Large White pigs.

[0060]

[0061] Note: Different letters in the same row of numbers indicate significant differences (P<0.05).

Claims

1. The application of primer pairs for detecting SNP markers related to muscle shear force on porcine chromosome 8 in screening Large White pigs with low muscle shear force, characterized in that, The SNP marker is located at nucleotide rs332282301 on chromosome 8 of the pig in the international pig genome version 11.1 reference sequence, and has A / G polymorphism. The muscle shear force of individuals with GG and AG types is significantly lower than that of individuals with AA type.

2. The application according to claim 1, characterized in that, The upstream primer of the primer pair is SEQ ID NO: 2, and the downstream primer is SEQ ID NO:

3.

3. A method for screening large white pig populations with low muscle shear force, characterized in that... This includes detecting the genotype of the rs332282301 nucleotide site in pigs, and selecting individuals with the GG and AG nucleotide types at the rs332282301 nucleotide site as priority for breeding stock.

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